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Purification of cells from human peripheral blood

Purification of cells from human peripheral blood. Mononuclear cells: Lymphocytes + Monocytes Monocytes Polymorphonuclear leukocytes (PMNs) or granulocytes Platelets or thrombocytes. Monocytes. Cell number. Lymphocytes. Basophils. Neutrophils. Eosinophils. Erythrocytes. Density (g/ml).

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Purification of cells from human peripheral blood

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  1. Purification of cells from human peripheral blood • Mononuclear cells: Lymphocytes + Monocytes • Monocytes • Polymorphonuclear leukocytes (PMNs) or granulocytes • Platelets or thrombocytes

  2. Monocytes Cell number Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes Density (g/ml) 1.060 1.070 1.080 1.090 1.100 Density of human blood cells Polymorphonuclear leukocytes Mononuclear

  3. 600g 15 min MC 1.077 g/ml RBC PMNs Isolation of human mononuclear cells I Density barrier method

  4. Axis-Shield Density Gradient Mediafor isolation of human PBMCs • Lymphoprep™: 9.1% diatrizoate, 5.7% polysaccharide; 1.077 g/ml, 295mOsm; endotoxin <0.13 EU/ml • Nycoprep™ 1.077: 14.1% Nycodenz®, 0.44% NaCl, 5 mM Tricine-NaOH, pH 7.0; 1.077 g/ml, 295 mOsm; endotoxin < 0.13 EU/ml • Optiprep™ (endotoxin < 0.13 EU/ml) diluted with any suitable balanced salt solution or culture medium to give a 1.077 g/ml medium (C03)

  5. Separation of PBMCs on Lymphoprep™ 6 ml diluted blood on 3 ml of Lymphoprep™ Blood:saline 1:1 Blood < 12 h old

  6. Lymphoprep™ competitors • Ficoll-Paque (GE Health Care (ex Amersham and Pharmacia) • Histopaque 1077 (Sigma) • Not endotoxin tested • More expensive endotoxin-tested versions, e.g. Sigma’s Histopaque 1077 Hybrimax

  7. Lymphoprep™ queries (I) • Anticoagulant? • Not anticoagulant sensitive • Do I have to dilute the blood? • Whole blood less easy to layer; poorer % yields • Can increase sample:Lymphoprep™ ratio • Yes – but not recommended • Can I use a leukocyte-rich plasma? • Yes, OK to separate PBMCs and PMNs • Centrifugation at 4°C rather than room temperature? • Yes, but need to increase time by 5 min

  8. Lymphoprep™ queries (II) • Ficoll-Paque or Histopaque work better • Impossible – media have identical composition • Poor definition of PBMC band at interface • Poor layering technique and/or brake problem • Poor separation from erythrocytes/PMNs • Blood > 12 h old; clinical specimens • Can I use it for non-human blood? • Maybe primates and some ruminants • How can I remove the platelets?

  9. Blood diluted 1:1 with saline 800g for 15 min PBMCs Medium Plastic frit displaced LymphoprepTM upwards Lymphoprep™ Tube

  10. 10 ml blood saline 1500g PBMCs 30 min Plasma + Platelets PMNs + RBCs 1.25 ml OptiPrep Isolation of PBMCs by flotation - mixer method (C04)

  11. Problem with mixer method • Ratio of cells:plasma (haematocrit value) • Normal males: 42-54% • Normal females: 38-46% • Some clinical samples may have significantly lower values

  12. 5 ml whole blood 350g 15 min Platelets 5 ml of 1.063 g/ml Leukocytes + RBCs Removal (isolation) of platelets on a density barrier (C12)

  13. saline PBMCs 600g 1.077 g/ml 20 min PMNs Blood + OptiPrep 1.095 g/ml Plasma + RBCs + Platelets Isolation of platelet-free PBMCs (C05)

  14. Axis-Shield Density Gradient Mediafor the isolation of human PMNs from whole blood • Polymorphprep™ • 13.8% diatrizoate, 8.0% dextran 500 • Density = 1.113 g/ml • Osmolality = 460 mOsm

  15. Whole blood 5 ml 450-500g PBMCs 30-35 min at 20oC PMNs 5 ml Polymorphprep RBCs Separation of PBMCs and PMNs on Polymorphprep™

  16. 1 2 3 4 How does Polymorphprep™ work?

  17. Polymorphprep™ Separation

  18. Relative number 4 8 12 16 20 24 28 32 Cell vol (femtolitres x 0.1) Coulter STKR Analysis ofPBMCsandPolymorphs

  19. Polymorphprep – critical points • Must use fresh (<2 h old) whole blood • Anticoagulant must be EDTA (dipotassium) • Temperature must be 18-22C • Blood:medium volume ratio must be 1:1 • 5 ml + 5 ml in a 15 ml tube • Brake must NOT be used • Centrifugation time can be + 5 min • Recommended g-force is the gav

  20. axis of rotation rmax rav rmin Geometry of rotors Set rpm speed to give 500g at rmax Equivalent to 400g at rav

  21. Polymorphprep/sample volumes

  22. 600g 20 min 1.077 g/ml 1.10 g/ml Human PMNs from a leukocyte-rich plasma (C011) PBMCs PMNs

  23. Axis-Shield Density Gradient Mediafor the isolation of human monocytes: barrier sedimentation (C55) • 13.0% Nycodenz, 0.58% (w/v) NaCl, 5 mM Tricine-NaOH, pH 7.0 • Density = 1.068 g/ml • Osmolality = 335 mOsm

  24. Monocytes Cell number Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes Density (g/ml) 1.060 1.070 1.080 1.090 1.100 Density of human blood cells

  25. LRP 600g 15 min Harvest zone M L 1.068 g/ml Separation of monocytes on 335 mOsm, 1.068 g/ml barrier

  26. Nycoprep™ 1.068 requirements • Leukocyte-rich plasma; not whole blood • Anticoagulant: EDTA • Blood from normal individual, < 2 h old • Room temperature 18-22°C • No brake to decelerate rotor • Centrifugation conditions may need optimizing from lab to lab

  27. HBS M 600-800g 1.068 g/ml 15-20 min L 1.084 g/ml P LC LRP Flotation of monocytes from leukocyte-rich plasma (C09)

  28. FACS analysis of monocyte bandGraziani-Bowering, G.M. et al (1997)J. Immunol. Meth., 207, 429-436

  29. Monocyte flotation isolation queries • No monocyte band observed • Band may be quite diffuse • Poor recovery and purity of monocytes • Rapid preparation of leukocytes essential and handling of cells must be very gentle • Does method work at 4°C? • Probably

  30. Axis-Shield Density Gradient Media:isolation of rodent PBMCs, sedimentation on to a barrier (C43) • Dilute buffered saline with water (2.5:0.5) Solution is 242 mOsm • Dilute OptiPrep with the 242 mOsm solution (2.7: 9.3) • Solution of 1.077 g/ml and 265 mOsm • Competition (Histopaque 1.083): 1.083 g/ml, 295 mOsm

  31. Monocytes Cell number Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes Density (g/ml) 1.060 1.070 1.080 1.090 1.100 Axis-Shield Density Gradient Mediafor the isolation of rodent PBMCs

  32. Isotonic ammonium chloride Aspirate 600g 20 min LRP MC 1.077 g/ml 265 mOsm RBC + PMNs Rodent PMNs from leukocyte-rich plasma (C45)

  33. Common flotation strategy for purifying low density cells from tissues • Isolation of dendritic cells from spleen, thymus, lymph nodes etc (C20) • Tissue disaggregated using enzymes • Cell suspension adjusted to 1.085 g/ml • Make up solution of 1.065 g/ml

  34. Saline or culture medium Dendritic cells 600g 1.065 g/ml 15 min Sample 1.085 g/ml Dendritic cells – flotation strategyReudl, C. et al (1996) Eur. J. Immunol., 26, 1801-1806

  35. Islets 1.09 g/ml 500g/5 min 1.10 g/ml Acinar cells Pancreatic islets – flotation (C18) Van der Burg, P.P.M. (1998) Transpl. Proc. 30, 362-363

  36. Viable cells Culture medium 1.12 g/ml 800g 20-25 min Cell suspension adjusted to 1.15-1.16 g/ml Removal of non-viable cells (C13)

  37. Other Axis-Shield Cell Applications • Epithelial cells from gastric mucosa (C28) • Neurons: spinal cord (C22) brain (C29) • Human erythrocytes and reticulocytes (C35) • Megakaryocytic progenitor cells (C23/C48) • Pneumocytes and other lung cells (C25/C44) • Stellate cells from liver and pancreas (C33) • Hepatic Kupffer cells (C50) • Renal cells (C42)

  38. Publications database on cells • OptiPrep (since 1994) over 650 • Nycodenz® (since 1984) approx 2000 • Using either the Applications CD or the website: • www.axis-shield-density-gradient-media.com • Follow the instructions to access the relevant Index • Click on the cell of interest

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